There is an urgent unmet need to develop targeted therapeutic options for Pancreatic Ductal Adenocarcinoma (PDAC), which continues to be a therapy recalcitrant disease. Data has shown that perturbation of two specific genes, K-Ras and CDKN2A, are nearly universal in PDAC. Based on this mutational profile, PDAC was thought initially to be a good candidate for CDK4 inhibition therapy. However, most PDAC cell lines and PDX models are resistant and this appears to be due to the activation of another kinase CDK2. The CDK4/6 specific inhibitors (CDK4i) are approved for metastatic HR+ breast cancer. However, while CDK4i therapy initially increases progression-free survival in ER+ metastatic breast cancer (BC) patients, ultimately the majority develop secondary resistance, due to compensation by CDK2. Thus, both CDK4i-refractory breast cancer and PDAC appear resistant by the same mechanism. Therefore, to generate a better inhibitor for metastatic breast cancer patients and PDAC, one needs to inhibit both the kinase driving cancer (CDK4/6) and the kinase causing resistance (CDK2). CDK2 small molecule inhibitors also target the highly conserved and essential CDK1 kinase and have met with unacceptable toxicity in the clinic, so this approach has proven unsuccessful. In this exploratory R21 application, we propose using a novel strategy. Instead of targeting the conserved active sites of CDK4/6 and CDK2, we target p27Kip1, which is a CDK assembly factor and CDK ON/OFF switch. Tyrosine (Y) phosphorylation of p27 (pY88) by the tyrosine kinase Breast tumor Related Kinase (Brk) causes a conformational change in p27 bound to CDKs, turning them ?ON?. We demonstrated that in breast cancer cells blocking phosphorylation of p27 with the therapeutic peptide ALT inactivated CDK4/6 and CDK2 and represented a new way to block ER+ BC cell proliferation, induce senescence and ultimately cause cell death, which translates into tumor regression and increased OS in BC animal models. We have shown that p27 is Y phosphorylated in several established PDAC cell lines, and expression of ALT in two cell lines inhibited proliferation, suggesting that CDK2 might be inhibited by ALT-mediated targeting of p27 in PDAC. Our hypothesis is that inhibition of CDK4/6 and CDK2 by the therapeutic peptide ALT will block proliferation of PDAC and represents a novel way to target this tumor type. Our specific Aims: 1) To examine the effect that ALT and pY blockage therapy has on PDAC cell lines in vitro. 2) To examine the therapeutic effect pY blockage therapy and ALT in vivo in PDAC mouse models. A panel of primary patient derived Cell (PDC) lines, which have been sequenced and characterized, and cross-matched PDX models will be used to test ALT's effect on cell growth and tumor progression. ALT will be delivered by two modes: inducible expression and a novel liposomal-peptide formulation. This exploratory R21 project will test the hypothesis that targeting p27 in clinically relevant in vitro and in vivo models, as a proof of concept approach, may represent a viable clinical strategy, which should be further explored. !